The purpose of this study was to investigate species specific inhibitory effects of esDNA isolated from two conspecific organisms: Vibrio parahaemolyticus (VP) and Vibrio harveyi (VH), and to assess the functional role of esDNA to enhance the survival rate of Artemia sp. In an in vitro study, nine doses of Extracellular self-DNA of Vibrio parahaemolyticus (esDNAVP) and Vibrio harveyi (esDNAVH) were used as the target for the challenge test with the conspecific bacteria. In an in vivo study, the protective effect of esDNA was then tested in nauplii of the brine shrimp Artemia at various priming times and concentrations of esDNA under gnotobiotic conditions prior to challenge with VP and VH at the concentration of 5 × 105 CFU mL-1. The results from in vitro study showed that the use of esDNAVP at levels of 24.02 and 48.05 ng µl-1 and esDNAVH at concentrations of 13.33 and 26.67 ng µl-1 were able to inhibit the growth of the conspecific species when added to the culture medium at the concentration level of 5 × 105 CFU mL-1. The results from in vivo study showed that the use of 24.02; 48.05 and 72.07 ng µl-1 of esDNAVP as well as the use of 13.33; 26.67 and 40.00 ng µl-1 of esDNAVH inhibited the growth of VP and VH and enhanced the survival rate of Artemia sp compared to the control treatment (P<0.05). Taken together, we confirmed that esDNA obtained from the extraction and random fragmentation from esDNAVP and esDNAVH, produces a species-specific inhibitory effect on the same species and can serve as a potential alternative strategy for disease control to deliver the functionality of esDNA to the fish and shrimp.

1. PRE-LIMINARY REPORT OF SELF – DNA IN AQUACULTURE:
NOVEL APPROACH TO INHIBIT THE GROWTH OF Vibrio sp
Presented By :
Dr. ROMI NOVRIADI, M.Sc
POLITEKNIK AHLI USAHA PERIKANAN
ONE HEALTH – NEW ROADS TOWARDS ONE HEALTH IN INDONESIA AND THE
REGION
THROUGH NO SELF MECHANISMS
IPB CONVENTION CENTER – SEPTEMBER 22, 2022

2. INDONESIA has
more than 17,500 islands
with at least 108,000 km² coastline
275,77 miliion people and the majority live in coastal areas
12,1 million tonnes for fisheries production
3,36 million ha for mangroves
Need to balance the ecological and economical
sustainability
"Ocean, sea, strait and bay are
the future of our civilization,"
Indonesia as world’s maritime axis
MISI
Kedaulatan
(Sovereignty)

3. Species 2015 2016 2017 2018 2019 2020
Milk fish 672.196 747.445 701.427 875.592 824.239 811.883
Giant gouramy 113.407 132.334 234.084 173.345 190.389 152.669
Others 344.128 591.197 590.945 358.249 343.368 311.331
Asian sebass 6.558 7.890 8.432 9.583 7.228 5.418
Groupers 16.795 11.504 70.294 16.414 13.829 9.478
Cat fishes 719.619 764.797 1.125.526 944.779 1.088.945 993.768
Common carp 461.546 497.208 316.649 534.215 620.831 560.669
Nile tilapia 1.084.281 1.114.156 1.280.126 1.171.236 1.317.561 1.172.633
Pangasius/catfish 339.069 392.918 319.967 373.262 380.130 327.146
Shrimps 607.152 692.568 919.988 911.857 863.119 881.599
Total 4.364.751 4.952.018 5.567.439 5.368.532 5.649.639 5.226.594
Indonesian Aquaculture Production Volume 2015-2020
AQUACULTURE PRODUCTION
1. Aquaculture – or fish farming – will provide close to two
thirds of global food fish consumption by 2030 as catches
from wild capture fisheries level off (World Bank)
2. Aquaculture, not the Internet, represents the most promising
investment opportunity of the 21st Century (Peter Drucker -
Management Expert & Economist)

4. Challenge To Optimize the Productivity

5. “Among infectious diseases, Vibriosis cause serious losses
in aquaculture"
1. Diseases reported as a significant challenge to the expansion of aquaculture production
2. Economic losses > $ 100 millions
3. High numbers of farms closed and large numbers of job losses
4. Will take years to recover
Number of Vibrio spp. on normal ponds and white feces infected ponds
Vibrio species %
V. vulnivicus 80
V. fluvialis 44
V. parahaemolyticus 26
V. alginolyticus 20
V. damselae 18
V. mimicus 8
V. Cholerae (non01) 6

6. Current Practices to Managing Vibriosis
1. Prophylactic approach
- Immunostimulation, vaccination,
probiotic, Quorum quenching?
2. Biosecurity
- Specific pathogen free and resistance
- Biosecurity devices, disinfection and
surveillance
3. Feeding Management
Avoid excess feeding – autofeeder?
4. Water quality management
- Waste management (nitrogen,
phosphorus and sulfide waste)
- Control of Vibrio and blue green algae
Culture system
• Biofloc
• Semi-biofloc
• Autotroph
• Heterotroph
• Recirculation
• Water exchange
• Zero water exchange
• Colloid
• Synbiotic
• Cultured probiotic
Microorganism

7. Self DNA: a mechanism to inhibit the growth of Vibrio sp
1. The extracellular structures may fulfill relevant roles in terms of structure and functional
organization, contributing to fundamental processes like cell adhesion, migration, proliferation,
differentiation, and apoptosis.
2. Extracellular structures can act as protective barriers in preventing pathogen invasion, or
represent advantageous habitats to facilitate symbiotic interactions, for example favoring
adhesion of microbial communities
Goal of the research:
1. To asses species-specific inhibitory effects of extracellular DNA from Vibrio harveyi and Vibrio
parahaemolyticus
2. To test the species-specific inhibitory effects of extracellular DNA to the growth of Vibrio sp in
brine shrimp Artemia sp as the important live food in aquaculture (Axenic condition)

8. Material and Methods
1. Bacterial Culture
The bacterial isolates used in this study were BT1H (Vibrio harveyi), GD24 (Vibrio
parahaemolyticus), and ASO2 (Vibrio parahaemolyticus). The isolates were obtained
from the Department of Aquaculture, Faculty of Agriculture, Gadjah Mada University.
2. DNA Extraction
DNA extraction was carried out manually using TNES (Tris NaCl EDTA Solution) and PCIAA
(Phenol Chloroform Isoamyl Alcohol) solutions
3. Electrophoresis of DNA Extraction
The DNA extraction results were then visualized by electrophoresis. The agarose gel
electrophoresis was used as an intermediate medium for DNA migration. The
concentration of agarose used in this stage was 1% by mixing 1 mg of agarose dissolved
in 15 ml of solvent solution (TBE 1x).

9. Material and Methods
4. Sonication
Sonication of the extracted DNA
fragments was carried out using an
ultra-sonicator machine (Ultrasonic
Generator US-300T, Nissei, Japan)
which functions to fragment the
DNA using ultrasonic waves to cut
the DNA into small sizes. The
sonication process was used to
fragment the DNA from 10,000 bp to
100 bp.
The agarose electrophoresis gel was then employed
to confirm the length of DNA and NanoDrop
spectrophotometer were used to quantify the
amount of DNA within the 100 bp.

10. Focus on Vibrio harveyi and Vibrio parahaemolyticus
V. parahaemolyticus I : >10.000 bp
V. parahaemolyticus II : >10.000 bp
V. alginolyticus : >10.000 bp
V. harveyii : > 10.000 bp
V. parahaemolyticus I : 100 bp
V. parahaemolyticus II : 100 bp
V. alginolyticus : 100 bp
V. harveyii : 100 bp
Sonication DNA process cut the length of Vibrio harveyi and
Vibrio parahaemolyticus from 10,000 bp to 100 bp

11. In-vitro test
1. The in-vitro test was performed in
a micro-plate filled with 100 µl
liquid zobell medium using 8 rows
of well
2. First row was filled with sonicated
DNA with concentration of 96.10
ng/µl for Vibrio parahaemolyticus
and 53.33 ng/µl for Vibrio harveyi
(Based on NanoDrop
measurement) – the final
concentration become 48.05
ng/µl for Vibrio parahaemolyticus
and 26.67 ng/µl for Vibrio harveyi
100 µL 100 µL 100 µL 100 µL 100 µL 100 µL 100 µL 100 µL
DNA concentration going to decrease
Figure. Dilution process of the sonicated DNA

12. In-vitro test using Vibrio parahaemolyticus
0
200
400
600
800
1000
1200
1400
0 48.05 24.02 12.01 6.01 3 1.5 0.75 0.37
Amount
of
V.
parahaemolyticus
(10
6
CFU/mL)
Concentration of self-DNA (ng/µL)
D
A
C
B
C C C
C BC

13. In-vitro test using Vibrio harveyi
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
1,000.00
0 26.67 13.34 6.67 3.34 1.67 0.84 0.42 0.21
Amount
of
V.
harveyi
(
10
6
CFU/mL)
Concentration of self-DNA (ng/µL)
B
A
AB
B B B B B B

14. 1
2
3
4
5
6
7
8
In-vitro test – Visual observation
Figure. Determination of inhibitory concentration for
self-DNA. Column A = Vibrio parahaemolyticus (positive
control); Column B = Vibrio parahaemolyticus + self-
DNA; Column C = Vibrio harveyi (positive control) and
Column D = Vibrio harveyi + self-DNA
In principle, the active living cells cause
reduction of resazurin from purple blue to
resofurin (pink-colorless). Color changes in
resazurin are carried out by enzymes in cells
in the mitochondria. In this assay, the
inhibitions were determined through
recording of the colour change observed.
This means that the inhibition ability of the
fragmented DNA will inhibit the growth of
the pathogen and the resazurin colour will
remains blue.

15. In-vivo test – Interaction Artemia and Vibrio sp
1. Artemia play an important role in aquaculture, as the primary feed during the larval stage
2. Non-selective filter feeder and Possible vector for diseases, including Vibriosis
• Creating gnotobiotic cultures starting with axenic larvae (Instar II nauplii)
 Only bacteria added to cultures are present!

16. Artemia culture in Axenic condition
Axenic condition = Free from other living organisms
Decapsulated Artemia
Immersed in self-DNA
Vibrio parahaemolyticus
48.05 ng/µl
Vibrio harveyii
26.67 ng/µl
Challenge test and Total Plate Count

17. Artemia challenge test - Results
0%
20%
40%
60%
80%
100%
120%
0 h 12 h 24 h 36 h 48 h 60 h
Observation Time
Survival
Rate
(SR)
V. harveyii (control)
V. harveyii + s-DNA
Challenge test with Vibrio parahaemolyticus at density of
5 x 105 cells/mL
Challenge test with Vibrio harveyi at density of
5 x 105 cells/mL
0%
20%
40%
60%
80%
100%
120%
0 h 12 h 24 h 36 h 48 h 60 h
Observation Time
Survival
Rate
(SR)
V. parahaemolyticus
(control)
V. parahaemolyticus +
s-DNA

18. Density of bacteria during the challenge test
-20
0
20
40
60
80
100
120
140
160
180
200
12 h 24 h 36 h 48 h
Observation Time
Ammount
of
V.
harveyii
(CFU/ml)
V. harveyii (control)
V. harveyii + s-DNA
0
20
40
60
80
100
120
140
160
180
200
12 h 24 h 36 h 48 h
Observation Time
Ammount
of
V.
parahaemolyticus
(CFU/ml)
V. parahaemolyticus (control)
V. parahaemolyticus + s-DNA

19. Conclusion
1. In the present study, certain concentration of self-DNA could inhibit the
growth of Vibrio parahaemolyticus and Vibrio harveyi
2. Based on in-vitro and in-vivo study, self-DNA could act as an alternative
strategy to minimize the Vibriosis incidence in invertebrate
3. Future study: Finfish with innate immune responses?
4. Quorum quenching properties?
• Now: bacteria sense and respond to
environment and to each other
o Extracellular signal molecules
o ≈ hormones in higher organisms
AHA!

20. Thank you to our collaborators:
novriadiromi@yahoo.com